Background[unreadable] [unreadable] During the last several years we have studied five different dominantly-inherited autoinflammatory/ autoimmune disorders. The first of these illnesses is the TNF receptor-associated periodic syndrome (TRAPS), which is characterized by prolonged attacks of fever, serositis, migratory rash and myalgia, arthritis, periorbital edema, conjunctivitis, and, in some patients, systemic amyloidosis. In 1999 we identified the first mutations in the gene encoding the 55 kDa tumor necrosis factor receptor (TNFRSF1A) in families with dominantly-inherited recurrent fevers, and proposed the name TRAPS for this clinical condition. Initial mechanistic studies indicated a defect in the activation-induced shedding of the p55 (but not p75) TNF receptor, possibly leading to impaired immune homeostasis. [unreadable] [unreadable] In 2002 we discovered dominantly-inherited de novo mutations in a second gene, CIAS1 (also known as NALP3 or PYPAF1), in about 50% of patients with a disorder known as neonatal onset multisystem inflammatory disease (NOMID) or chronic infantile neurologic cutaneous and articular (CINCA) syndrome. Manifestations of NOMID/CINCA may include daily fevers, an urticaria-like skin rash, chronic aseptic meningitis, uveitis, papilledema, sensorineural hearing loss, mental retardation, patellar and epiphyseal long bone overgrowth, and systemic amyloidosis. Two milder conditions, familial cold autoinflammatory syndrome (FCAS) and Muckle-Wells syndrome (MWS), are caused by mutations in the same gene. CIAS1 encodes a protein, cryopyrin, that participates in a macromolecular complex called the inflammasome to regulate the activation of interleukin-1 (IL-1) beta. Collectively, all three diseases are known as the cryopyrin-associated periodic syndromes (CAPS).[unreadable] [unreadable] A fifth dominantly-inherited autoinflammatory disorder, denoted the syndrome of pyogenic arthritis with pyoderma gangrenosum and acne (PAPA), is caused by mutations in a protein known as proline serine threonine phosphatase interacting protein (PSTPIP1). PAPA is characterized by episodes of sterile pyogenic arthritis, which can be destructive if not treated, formation of open, purulent ulcers of the skin (pyoderma gangrenosum), and severe cystic acne. In 2003 our group discovered that PSTPIP1 binds pyrin, the protein mutated in familial Mediterranean fever (FMF), and that disease-associated mutations in PSTPIP1 lead to more avid binding to pyrin, and increased IL-1 beta activation, relative to healthy controls.[unreadable] [unreadable] Results of the Last Year[unreadable] [unreadable] Functional studies of mutant TNFRSF1A molecules: In collaboration with the laboratory of Dr. Richard Siegel, we have continued analyses of the functional consequences of TRAPS-associated TNFRSF1A mutations. Studies performed during previous reporting periods have demonstrated that the aforementioned defect in TNF receptor ectodomain cleavage is unlikely to explain all or most of the autoinflammatory phenotype, and in transfection systems the mutant receptors have been found to exhibit diminished binding to TNF and abnormal intracellular trafficking, with retention in the endoplasmic reticulum (ER). During the present reporting period we have continued mechanistic studies in primary patient leukocytes, TRAPS knockin mice, and cell lines, and have recently submitted a manuscript summarizing our findings. In this manuscript we demonstrated that mutant TNFRSF1A protein accumulates intracellularly in knockin mice with two independent TRAPS-associated TNFRSF1A mutations and in TRAPS patient peripheral blood mononuclear cells. Cells harboring TRAPS-associated TNFRSF1A mutations exhibited spontaneous enhanced p38 and JNK MAP-kinase activation in response to LPS, while NF-kappa B activation was not affected. These signaling abnormalities led to increased production of pro-inflammatory cytokines in immune cells from knockin mice and TRAPS patients. Heterozygous TRAPS knockin mice exhibited hypersensitivity to LPS, while homozygous TNFRSF1A mutant mice had a phenotype identical to TNFRSF1A knockout mice. Hypersecretion of cytokines by heterozygous TNFRSF1A mutant cells was dependent on reactive oxygen species (ROS), most likely generated by NADPH oxidase (NOX) enzymes. These results define a pathway of MAPK and ROS-dependent inflammation triggered by intracellularly accumulated TNFRSF1A in TRAPS. Full expression of the inflammatory phenotype in TRAPS depends on the wild-type TNFRSF1A, although the wild-type and mutant receptors do not physically associate. Inhibiting ROS may synergize with TNF blockade in the treatment of this and potentially other autoinflammatory diseases.[unreadable] [unreadable] Gene expression profiling in cryopyrin-associated periodic syndromes (CAPS): We have continued studies begun in the previous reporting period concerning gene-expression profiling in CAPS patients before and after treatment with the IL-1 receptor antagonist, anakinra, and compared with healthy controls. CAPS patients respond dramatically to anakinra, consistent with a central role for IL-1 in clinical manifestations. To understand better the pathogenesis of CAPS we compared gene expression patterns in peripheral blood mononuclear cells (PBMCs) from 22 CAPS patients and 14 healthy children. We also collected 16 paired samples from CAPS patients before and after anakinra to identify genes that are highly responsive to IL-1beta inhibition. Despite stringent data analysis many highly differentially expressed genes were found in both data sets. Quantitative RT-PCR was performed on selected differentially expressed genes to confirm the microarray data. We validated several genes that are involved in regulation of reactive oxygen species (ROS) including p22phox, p40phox, TXN, and TXNIP. Treatment of PBMCs from CAPS patients and healthy controls with DPI, an inhibitor of NADPH oxidase, showed that ROS signaling was necessary for IL-1beta production in PBMC. A gene expression-based model was generated using a subset of 16 CAPS samples and 66 non-CAPS samples (including healthy controls and individuals with other autoinflammatory disorders) to differentiate CAPS patients from controls and other patients with systemic inflammation. The CAPS-specific gene expression model correctly classified 6/6 independent pre-anakinra CAPS samples and 11/11 independent non-CAPS samples. This classifier also correctly identified 15/16 post-anakinra CAPS samples despite the fact that the CAPS patients were in clinical remission. [unreadable] [unreadable] Studies of IL-1 inhibition in the cryopyrinopathies: In collaboration with Dr. Raphaela Goldbach-Mansky, we continue therapeutic trials of anakinra and a longer-acting IL-1 inhibitor manufactured by Regeneron pharmaceuticals and denoted IL-1 Trap. These studies are more thoroughly presented under project Z01 AR041138-06 OCD.[unreadable] [unreadable] Generation of new animal models: In collaboration with Dr. Hal Hoffman, we have engineered mice carrying the Muckle-Wells syndrome mutation, A352V. Pups expressing this mutation show profound growth retardation, hair loss, skin scaling, and die about ten days after birth. Massive neutrophilic influxes in multiple tissues, as well as intense in situ staining for IL-1beta and IL-6, suggest an uncontrolled inflammatory reaction. In culture, macrophages from knockin mice secrete IL-1beta in response to LPS, but do not require secondary stimulation with ATP as is the case for cells from wild-type mice. IL-1beta appears to be the main cytokine driving the disease process in these mice, in that knockins bred onto an IL-1beta receptor knockout background are disease-free. Treatment with the IL-1beta inhibitor, IL-1 Trap, extends the lives of knockin mice by about four days.[unreadable] [unreadable] We have also generated PSTPIP1 knockout mice, and the PSTPIP1 E250Q PAPA knockin mouse. Studies to characterize the phenotype of these mice are now under way.